The amount of each bacterial species and of eubacteria was evaluated by qPCR using specific primers (Life Technologies) and TaqMan probes (Life Technologies or Roche; Table S1). First, DNA was purified with the QIAamp DNA Mini Kit (Qiagen GmbH) following the manufacturer’s instructions for buccal swabs. Then, reactions were performed as previously described [20 (link)], and the resulting crossing points were transformed to number of copies/µl through plasmid standard curves. Each standard plasmid was prepared by cloning the target PCR products in pGEMT [21 ] with the specific primers from Table S1, using DNA from E. brachy DSM 3990, F. alocis ATCC 35,896, F. fastidiosum DSM 25,557, P. endodontalis ATCC 35,406, P. gingivalis ATCC 33,277, S. sputigena ATCC 35,185, T. forsythia ATCC 43,037, T. denticola DSM 14,222 and T. socranskii ATCC 35,534 as templates. The number of copies/µl was estimated using a Nanodrop ND-1000 UV – vis spectrophotometer (Nanodrop Technologies).
Eubacterial load was used to normalise the data between individuals. The standard curve was prepared as in Àlvarez et al. [20 (link)], using DNA from Streptococcus gordonii ATCC 49,818, Veillonella parvula NCTC 11,810, Actinomyces naeslundii DSM 17,233, Fusobacterium nucleatum DSM 20,482 and P. gingivalis ATCC 33,277 as templates.
Eubacterial load was used to normalise the data between individuals. The standard curve was prepared as in Àlvarez et al. [20 (link)], using DNA from Streptococcus gordonii ATCC 49,818, Veillonella parvula NCTC 11,810, Actinomyces naeslundii DSM 17,233, Fusobacterium nucleatum DSM 20,482 and P. gingivalis ATCC 33,277 as templates.
